Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

Abstract

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

ACKNOWLEDGMENTS

We thank Elke Fenner for technical assistance, H. Küster for providing pGUS-INT, Y. D'Aubenton-Carafa and C. Thermes for RNA structure analysis and helpful discussions, and L. Troussard for sequencing help.

C.J. was supported by a postdoctoral fellowship from the Swedish Council for Forestry and Agricultural Research and an EEC PTP training fellowship, C. Sousa by a postdoctoral fellowship from the EEC (TMR Marie Curie training grant), C.C. by a fellowship from the Ministère Français de l'Enseignement Supérieur et de la Recherche, and H.M. by a short-term EMBO fellowship.